| 王元欣,张 磊,岳康异,黑 悦,鱼 洋,武秀权,蒋晓帆.SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义[J].,2018,(5):817-821 |
| SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义 |
| The Expression and Significance of SIRT3 in Mouse Cortical Neurons after Oxygen - glucose Deprivation/Reperfusion |
| 投稿时间：2017-09-21 修订日期：2017-10-18 |
| DOI：10.13241/j.cnki.pmb.2018.05.004 |
| 中文关键词: SIRT3 LC3 Cleaved caspase-3 自噬 凋亡 |
| 英文关键词: SIRT3 LC3-II Cleaved caspase-3 Autophagy Apoptosis |
| 基金项目:国家自然科学基金项目(8167050366) |
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| 中文摘要: |
| 摘要 目的：通过建立体外脑缺血模型，探讨沉默信息因子3（SIRT3）在小鼠皮层神经元氧糖剥夺再灌注（OGD/R）损伤后的表达和意义。方法：C57BL/6J小鼠皮层神经元原代培养7天后，以氧糖剥夺不同时长（2 h、4 h、6 h、8 h）再灌注24 h作为观察时间点，利用细胞增殖-毒性检测试剂盒（Cell Counting Kit-8, CCK-8）检测细胞活力；小鼠乳酸脱氢酶（LDH）试剂盒检测LDH释放；蛋白印迹法（Western blot WB）观察微管相关蛋白1轻链3（LC3-Ⅱ）、活化凋亡蛋白3（Cleaved caspase-3）、以及SIRT3的表达变化；免疫荧光下进一步观察LC3-II、SIRT3表达。结果：与正常组比，随着氧糖剥夺时间的延长，LDH释放量呈台阶式升高（P <0.01），而神经元活性进展性下降（P <0.01）；蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调，并于OGD 4h 达峰值，SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状，而Cleaved caspase-3整体上调；相应的，细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达，与此同时SIRT3荧光强度亦增高。结论：神经元缺血时间越长损伤越重；LC3-Ⅱ和SIRT3表达呈现相似性；SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。 |
| 英文摘要: |
| ABSTRACT Objective: To verify the expression and significance of silent information regulator 3 (SIRT3) in cerebral ischaemia using the oxygen glucose deprivation/reperfusion (OGD/R) model in vitro. Methods: Primary cultured cortical neurons from C57BL/6J exposed to oxygen-glucose deprivation for 2 h, 4 h, 6 h, 8h , and followed by 24 h of reperfusion. Additionally, the cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, the cytotoxicity was measured by LDH kit assay, the expression of LC3-II, cleaved caspase-3 and SIRT3 was determined by western blot and the levels of LC3-II and SIRT3 were further asssayed by immunofluorescence. Results: The results showed that there was a time-dependent increase of LDH release and decrease of cell viability (P<0.01). The results of Western blot demonstrated that LC3-II and SIRT3 increased and peaked at OGD 4h while the expression of Cleaved caspase-3 was elevated all the time. The results of immunofluorescence indicated that LC3-II and SIRT3 were significantly expressed in somas and neurites compared with the control. Conclusion: We observed that the expression of LC3 and SIRT3 changes in a similar pattern through a OGD/R model. Hence, SIRT3 might protects neurons against ischemic injury via regulating autophagy. |
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